| 1. |
- Schütz, Patrick, et al.
(författare)
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Comparative structural analysis of human DEAD-box RNA helicases
- 2010
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 5:9
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Tidskriftsartikel (refereegranskat)abstract
- DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.
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| 2. |
- Abou-Hamdan, Abbas, et al.
(författare)
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Functional design of bacterial superoxide : quinone oxidoreductase
- 2022
-
Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1863:7
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Tidskriftsartikel (refereegranskat)abstract
- The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions. It can perform a range of cellular functions, including bacterial warfare and iron uptake, signalling and host immune response in eukaryotes. However, it also serves as precursor for more deleterious species such as the hydroxyl anion or peroxynitrite and defense mechanisms to neutralize superoxide are important for cellular health. In addition to the soluble proteins superoxide dismutase and superoxide reductase, recently the membrane embedded diheme cytochrome b561 (CybB) from E. coli has been proposed to act as a superoxide:quinone oxidoreductase. Here, we confirm superoxide and cellular ubiquinones or menaquinones as natural substrates and show that quinone binding to the enzyme accelerates the reaction with superoxide. The reactivity of the substrates is in accordance with the here determined midpoint potentials of the two b hemes (+48 and -23 mV / NHE). Our data suggest that the enzyme can work near the diffusion limit in the forward direction and can also catalyse the reverse reaction efficiently under physiological conditions. The data is discussed in the context of described cytochrome b561 proteins and potential physiological roles of CybB.
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| 3. |
- Andersson, Martin E, et al.
(författare)
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Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2.
- 2004
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Ingår i: Biochemistry. - 0006-2960. ; 43:24, s. 7966-72
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Tidskriftsartikel (refereegranskat)abstract
- The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.
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| 4. |
- Chaptal, Vincent, et al.
(författare)
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Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter
- 2022
-
Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 8:4
-
Tidskriftsartikel (refereegranskat)abstract
- Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg2+-bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when over-expressed in B. subtilis. Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.
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| 5. |
- Collins, Ruairi, et al.
(författare)
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Biochemical discrimination between selenium and sulfur 1 : a single residue provides selenium specificity to human selenocysteine lyase
- 2012
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Ingår i: PLoS One. - Stockholm : Karolinska Institutet, Dept of Medical Biochemistry and Biophysics. - 1932-6203.
-
Tidskriftsartikel (refereegranskat)abstract
- Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.
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| 6. |
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| 7. |
- Karlberg, Tobias, et al.
(författare)
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Structure of human argininosuccinate synthetase.
- 2008
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Ingår i: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 64:Pt 3, s. 279-86
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Tidskriftsartikel (refereegranskat)abstract
- Argininosuccinate synthetase catalyzes the transformation of citrulline and aspartate into argininosuccinate and pyrophosphate using the hydrolysis of ATP to AMP and pyrophosphate. This enzymatic process constitutes the rate-limiting step in both the urea and arginine-citrulline cycles. Previous studies have investigated the crystal structures of argininosuccinate synthetase from bacterial species. In this work, the first crystal structure of human argininosuccinate synthetase in complex with the substrates citrulline and aspartate is presented. The human enzyme is compared with structures of argininosuccinate synthetase from bacteria. In addition, the structure also provides new insights into the function of the numerous clinical mutations identified in patients with type I citrullinaemia (also known as classic citrullinaemia).
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| 8. |
- Rollauer, Sarah E., et al.
(författare)
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Structure of the TatC core of the twin-arginine protein transport system
- 2012
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 492:7428, s. 210-
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Tidskriftsartikel (refereegranskat)abstract
- The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.
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| 9. |
- Shilova, Anastasiia, et al.
(författare)
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Current status and future opportunities for serial crystallography at MAX IV Laboratory
- 2020
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Ingår i: Journal of Synchrotron Radiation. - Chichester : Wiley-Blackwell. - 0909-0495 .- 1600-5775. ; 27, s. 1095-1102
-
Tidskriftsartikel (refereegranskat)abstract
- Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.
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| 10. |
- Ahlstrand, Tuuli, et al.
(författare)
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Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
- 2018
-
Ingår i: Virulence. - : Informa UK Limited. - 2150-5594 .- 2150-5608. ; 9:1, s. 1205-1223
-
Tidskriftsartikel (refereegranskat)abstract
- Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43nM in static settings and 2.4M in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested -lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.
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| 11. |
- Andersson, Charlotta S., 1979-, et al.
(författare)
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A dynamic c terminal segment in the mycobacterium tuberculosis mn/fe r2lox protein can adopt a helical structure with possible functional consequences
- 2012
-
Ingår i: Chemistry and Biodiversity. - : Wiley. - 1612-1872 .- 1612-1880. ; 9:9, s. 1981-1988
-
Tidskriftsartikel (refereegranskat)abstract
- Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.
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| 12. |
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| 13. |
- Andersson, Charlotta S, et al.
(författare)
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A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold
- 2009
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 106:14, s. 5633-8
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis R2c is the prototype for a recently discovered group of ribonucleotide reductase R2 proteins that use a heterodinuclear Mn/Fe redox cofactor for radical generation and storage. Here, we show that the Mycobacterium tuberculosis protein Rv0233, an R2 homologue and a potential virulence factor, contains the heterodinuclear manganese/iron-carboxylate cofactor but displays a drastic remodeling of the R2 protein scaffold into a ligand-binding oxidase. The first structural characterization of the heterodinuclear cofactor shows that the site is highly specific for manganese and iron in their respective positions despite a symmetric arrangement of coordinating residues. In this protein scaffold, the Mn/Fe cofactor supports potent 2-electron oxidations as revealed by an unprecedented tyrosine-valine crosslink in the active site. This wolf in sheep's clothing defines a distinct functional group among R2 homologues and may represent a structural and functional counterpart of the evolutionary ancestor of R2s and bacterial multicomponent monooxygenases.
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| 14. |
- Andersson, Charlotta Selina, 1979-
(författare)
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Structural studies of R2 and R2–like proteins with a heterodinuclear Mn/Fe cofactor and enzymes involved in Mycobacterium tuberculosis lipid metabolism
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tuberculosis is a notorious disease responsible for the deaths of 1.4 million people worldwide. A third of the world's population is infected with Mycobacterium tuberculosis, the bacterium causing the disease. The increase of multi drug-resistant strains worsens the situation, and the World Health Organization has declared tuberculosis to be a global emergency. The bacterium envelopes itself with a unique set of very long-chain lipids that play an important role in virulence and drug resistance. Therefore enzymes involved in lipid metabolism are putative drug targets. To allow entry into different metabolic pathways and transmembrane transport, fatty acids have to be activated. This is done primarily by fatty acyl-CoA synthetases (ACSs). We identified an ACS possibly involved in the bacterium’s virulence and solved its structure. Structural interpretation combined with previously reported data gives us insights into the details of its function. This enzyme is known to harbor lipid substrates longer than the enzyme itself, and we now propose how this peripheral membrane protein accommodates its substrates. Some of the most chemically challenging oxidations are performed by dinuclear metalloproteins belonging to the ferritin-like superfamily. We show that the ferritin-like protein, R2lox, from M. tuberculosis contains a new type of heterodinuclear Mn/Fe cofactor. This protein cofactor is capable of performing potent 2-electron oxidations as demonstrated by a novel tyrosine-valine crosslink observed in the protein. Recently a new subclass of ribonucleotide reductase (RNR) R2 proteins, was identified in the intracellular pathogen Chlamydia trachomatis containing the same type of Mn/Fe cofactor mentioned above. The RNR R2 proteins use their metal site to generate a stable radical, essential for the reduction of ribonucleotides to their deoxy forms, the building blocks of DNA. With this work, we were able to characterize the architecture of this metal cofactor.
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| 15. |
- Andersson, Charlotta S., 1979-, et al.
(författare)
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The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.
- 2012
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134:1, s. 123-125
-
Tidskriftsartikel (refereegranskat)abstract
- The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.
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| 16. |
- Andersson, Charlotta S., et al.
(författare)
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The Mycobacterium tuberculosis Very-Long-Chain Fatty Acyl-CoA Synthetase : Structural Basis for Housing Lipid Substrates Longer than the Enzyme
- 2012
-
Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 20:6, s. 1062-1070
-
Tidskriftsartikel (refereegranskat)abstract
- The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C-26). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.
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| 17. |
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| 18. |
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| 19. |
- Assarsson, Maria, et al.
(författare)
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Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.
- 2001
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:29, s. 26852-26859
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.
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| 20. |
- Atanassova, Anelia, et al.
(författare)
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High throughput methods for analyzing transition metals in proteins on a microgram scale.
- 2008
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Ingår i: Structural Proteomics. - 9781588298096
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Bokkapitel (refereegranskat)abstract
- Transition metals are among the most common ligands that contribute to the biochemical and physiological properties of proteins. In the course of structural proteomic projects, the detection of transition metal cofactors prior to the determination of a high-resolution structure is extremely beneficial. This information can be used to select tractable targets from the proteomic pipeline because the presence of a metal often improves protein stability and can be used to help solve the phasing problem in x-ray crystallography. Recombinant proteins are often purified with substoichiometric amounts of metal loaded, so additional metal may be needed to obtain the homogeneous protein solution crucial for structural analysis. Furthermore, identifying a metal cofactor provides a clue about the nature of the biological role of an unclassified protein and can be applied with structural data in the assignation of a putative function. Many of the existing methods for transition metal analysis of purified proteins have limitations, which include a requirement for a large quantity of protein or a reliance on equipment with a prohibitive cost.The authors have developed two simple high throughput methods for identifying metalloproteins on a microgram scale. Each of the techniques has distinct advantages and can be applied to address divergent experimental goals. The first method, based on simple luminescence and colorimetric reactions, is fast, cheap, and semiquantitative. The second method, which employs HPLC separation, is accurate and affords unambiguous metal identification.
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| 21. |
- Bennett, Matthew, et al.
(författare)
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Crystal structure of the essential biotin-dependent carboxylase AccA3 from Mycobacterium tuberculosis
- 2017
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 7:5, s. 620-626
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Tidskriftsartikel (refereegranskat)abstract
- Biotin-dependent acetyl-CoA carboxylases catalyze the committed step in type II fatty acid biosynthesis, the main route for production of membrane phospholipids in bacteria, and are considered a key target for antibacterial drug discovery. Here we describe the first structure of AccA3, an essential component of the acetyl-CoA carboxylase system in Mycobacterium tuberculosis (MTb). The structure, sequence comparisons, and modeling of ligand-bound states reveal that the ATP cosubstrate-binding site shows distinct differences compared to other bacterial and eukaryotic biotin carboxylases, including all human homologs. This suggests the possibility to design MTb AccA3 subtype-specific inhibitors.
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| 22. |
- Berntsson, Ronnie P. -A., et al.
(författare)
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Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:4, s. 2725-2735
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Tidskriftsartikel (refereegranskat)abstract
- The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.
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| 23. |
- Berthold, Catrine L., et al.
(författare)
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Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG
- 2009
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 106:34, s. 14247-14252
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Tidskriftsartikel (refereegranskat)abstract
- ADP-ribosylation is a ubiquitous regulatory posttranslational modi- fication involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dini- trogenase reductase-activating glycohydrolase (DraG), a dimanga- nese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme’s preference for manganese over magne- sium, although not requiring a redox active metal for the reaction.
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| 24. |
- Covarrubias, Adrian Suarez, et al.
(författare)
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Structural, biochemical and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 381:3, s. 622-633
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Tidskriftsartikel (refereegranskat)abstract
- Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C-gamma in the formation of the alpha beta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth, such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism.
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| 25. |
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| 26. |
- Diamanti, Riccardo, et al.
(författare)
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Comparative structural analysis provides new insights into the function of R2-like ligand-binding oxidase
- 2022
-
Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:12, s. 1600-1610
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Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.
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| 27. |
- Diamanti, Riccardo, 1988-
(författare)
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Shining light on unknown function proteins
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The study of protein structure and function is a fundamental step for understanding biological systems and for creating a solid ground for future drug development. Despite the development of novel ground-breaking biochemical and bioinformatical tools in the last decades, most proteins found in nature still have unknown assigned function. In this thesis, I present four publications aimed at protein structure and function determination. In these publications we present one method for high-throughput condition screening of membrane proteins for structural studies and three publications that aim at contributing to the understanding of proteins found in the pathogenic bacterium Mycobacterium tuberculosis, the deadliest bacterium worldwide, responsible for over 1 million deaths every year. Membrane proteins are very important targets for drug development. However, the study of membrane proteins is much more complex than fortheir soluble counterpart. In paper I, we developed a high-throughput method for detergent screening of membrane proteins that significantly reduces the time needed to find optimal conditions for structural studies. We developed amethod that allows for the screening of 192 protein-detergent conditions in 3 days. We used a TEV-folding reporter GFP-His tag vector to be able to measure the stability and amount of each sample after detergent solubilization of the cell lysate.In paper II, we characterized the Mycobacterium smegmatis respiratory supercomplex accessory proteins AscF and AscG. AscF and AscG arelocated in close proximity to the D and K proton pathways of Cytochrome c oxidase and might play a regulatory role in the respiratory supercomplex. Wes how how AscF and AscG do not bind nor metals nor nucleotides, but that they are essential for the normal growth of the M. smegmatis. We also show that both proteins are highly conserved in almost whole Mycobacteriales families, including M. tuberculosis. In paper III, we present two novel structures of the ferritin-like protein R2lox and further characterize the co-purified ligands in its lipid-binding pocket. We demonstrate how the two prevalent ligands that co-purify with the enzyme are fatty acids that host a C12 hydroxy-group and a double bond between the hydroxy group and the carboxylic group. The new structures from Sulfolobus acidocaldarius and Saccharopolyspora erythraea reveal new information about the enzyme’s possible regulatory mechanism and its possible interaction with partner proteins. In paper IV, we performed structural and functional studies of nicotinamide adenine dinucleotide (NAD+) synthase (NadE), which performs the last step of the de novo synthesis of NAD+: the amidation of nicotinic acidadenine dinucleotide (NaAD) into NAD+. We showed that some prokaryotes concert up to three NadE isoforms depending on external ammonium (NH3) availability. During high external concentrations of NH3, the ammonium-dependent NadE acts as primary catalyst of NAD+ synthesis. However, two glutamine-dependent homologues are capable of deamidation of glutamine when external ammonium availability is reduced. We show how the octameric glutamine-dependent NadE is the principal catalyst during nitrogen-fixing conditions, while the dimeric variant is the primary catalyst when NH3 availability is limited.
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| 28. |
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| 29. |
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| 30. |
- Grāve, Kristīne, 1988-, et al.
(författare)
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High-throughput strategy for identification of Mycobacterium tuberculosis membrane protein expression conditions using folding reporter GFP
- 2022
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 198
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacterium tuberculosis membrane protein biochemistry and structural biology studies are often hampered by challenges in protein expression and selection for well-expressing protein candidates, suitable for further investigation. Here we present a folding reporter GFP (frGFP) assay, adapted for M. tuberculosis membrane protein screening in Escherichia coli Rosetta 2 (DE3) and Mycobacterium smegmatis mc24517. This method allows protein expression condition screening for multiple protein targets simultaneously by monitoring frGFP fluorescence in growing cells. We discuss the impact of common protein expression conditions on 42 essential M. tuberculosis H37Rv helical transmembrane proteins and establish the grounds for their further analysis. We have found that the basal expression of the lac operon in the T7-promoter expression system generally leads to high recombinant protein yield in M. smegmatis, and we suggest that a screening condition without the inducer is included in routine protein expression tests. In addition to the general observations, we describe conditions allowing high-level expression of more than 25 essential M. tuberculosis membrane proteins, containing 2 to 13 transmembrane helices. We hope that these findings will stimulate M. tuberculosis membrane protein research and aid the efforts in drug development against tuberculosis.
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| 31. |
- Grāve, Kristīne, et al.
(författare)
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Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access
- 2019
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:6, s. 849-861
-
Tidskriftsartikel (refereegranskat)abstract
- Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y center dot) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 angstrom) and in complex with manganese (Mn-II/Mn-II, 1.30 angstrom). We also report three structures of the protein in complex with iron, either prepared anaerobically (Fe-II/Fe-II form, 1.32 angstrom), or prepared aerobically in the photo-reduced Fe-II/Fe-II form (1.63 angstrom) and with the partially oxidized metallo-cofactor (1.46 angstrom). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.
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| 32. |
- Grāve, Kristīne, 1988-
(författare)
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Structural basis for metalloprotein catalysis : Characterization of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase PgsA1 and Bacillus anthracis ribonucleotide reductase R2
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- About a third of all proteins need to associate with a particular metal ion or metallo-inorganic cofactor to function. This interplay expands the catalytic repertoire of enzymes and reflects the adaption of these catalytic macromolecules to the environments they have evolved in. A large portion of this work focuses on the membrane metalloprotein PgsA1 from the pathogen Mycobacterium tuberculosis and a radical-harboring protein R2 from the pathogen Bacillus anthracis, offering a glimpse into the metalloprotein universe and the catalysis they perform.This thesis is divided into two parts; the first part describes a method for high-throughput M. tuberculosis membrane protein expression screening in Escherichia coli and Mycobacterium smegmatis. This method employs target membrane protein fusions with the folding reporter Green Fluorescent Protein, allowing for fast selection of well-expressing membrane protein targets for further structural and functional characterization. This technique allowed overexpression of M. tuberculosis phosphatidylinositol phosphate synthase PgsA1, leading to its crystallization and the characterization of its high-resolution three-dimensional structure. PgsA1 is a MgII- dependent enzyme, catalyzing a vital step in the biosynthesis of phosphatidylinositol – one of the major phospholipids comprising the complex mycobacterial cell envelope. Therefore, PgsA1 presents an attractive target for the development of new antibiotics against tuberculosis.The second part of this thesis concerns the structural characterization of the B. anthracis class Ib ribonucleotide reductase radical-generating subunit R2 (R2b). R2b contains a dinuclear metallocofactor, which is able to be activated by dioxygen and generates a stable tyrosyl radical; the radical is further used for initiation of nucleotide reduction in the catalytic subunit of ribonucleotide reductase. R2b proteins utilize a di-manganese cofactor in vivo, but can also generate the radical using a di-iron cofactor in vitro, albeit less efficiently. How does R2b achieve correct metallation for efficient catalysis? We show that the B. anthracis R2b protein scaffold is able to select manganese over iron, and furthermore, describe the structural features that govern this metal-specificity. In addition, we describe redox-dependent structural changes in di-iron B. anthracis R2b after reaction with O2, and propose their role in gating solvent access to the metallocofactor and the radical site.
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| 33. |
- Grāve, Kristīne, et al.
(författare)
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Structure of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase reveals mechanism of substrate binding and metal catalysis
- 2019
-
Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 2
-
Tidskriftsartikel (refereegranskat)abstract
- Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9 angstrom), in complex with Mn2+ and citrate (1.9 angstrom), and with the CDP-DAG substrate (1.8 angstrom). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D-myo-inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism-including a substrate-induced carboxylate shift-for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life.
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| 34. |
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| 35. |
- Grāve, Kristīne, et al.
(författare)
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The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron
- 2020
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 25:4, s. 571-582
-
Tidskriftsartikel (refereegranskat)abstract
- Correct protein metallation in the complex mixture of the cell is a prerequisite for metalloprotein function. While some metals, such as Cu, are commonly chaperoned, specificity towards metals earlier in the Irving-Williams series is achieved through other means, the determinants of which are poorly understood. The dimetal carboxylate family of proteins provides an intriguing example, as different proteins, while sharing a common fold and the same 4-carboxylate 2-histidine coordination sphere, are known to require either a Fe/Fe, Mn/Fe or Mn/Mn cofactor for function. We previously showed that the R2lox proteins from this family spontaneously assemble the heterodinuclear Mn/Fe cofactor. Here we show that the class Ib ribonucleotide reductase R2 protein from Bacillus anthracis spontaneously assembles a Mn/Mn cofactor in vitro, under both aerobic and anoxic conditions, when the metal-free protein is subjected to incubation with Mn-II and Fe-II in equal concentrations. This observation provides an example of a protein scaffold intrinsically predisposed to defy the Irving-Williams series and supports the assumption that the Mn/Mn cofactor is the biologically relevant cofactor in vivo. Substitution of a second coordination sphere residue changes the spontaneous metallation of the protein to predominantly form a heterodinuclear Mn/Fe cofactor under aerobic conditions and a Mn/Mn metal center under anoxic conditions. Together, the results describe the intrinsic metal specificity of class Ib RNR and provide insight into control mechanisms for protein metallation.
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| 36. |
- Griese, Julia J., et al.
(författare)
-
Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase
- 2019
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:2, s. 211-221
-
Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O-2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O-2, but prefers manganese in the presence of O-2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O-2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O-2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.
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| 37. |
- Griese, Julia J., et al.
(författare)
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Assembly of nonheme Mn/Fe active sites in heterodinuclear metalloproteins
- 2014
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 19:6, s. 759-774
-
Forskningsöversikt (refereegranskat)abstract
- The ferritin superfamily contains several protein groups that share a common fold and metal coordinating ligands. The different groups utilize different dinuclear cofactors to perform a diverse set of reactions. Several groups use an oxygen-activating di-iron cluster, while others use di-manganese or heterodinuclear Mn/Fe cofactors. Given the similar primary ligand preferences of Mn and Fe as well as the similarities between the binding sites, the basis for metal specificity in these systems remains enigmatic. Recent data for the heterodinuclear cluster show that the protein scaffold per se is capable of discriminating between Mn and Fe and can assemble the Mn/Fe center in the absence of any potential assembly machineries or metal chaperones. Here we review the current understanding of the assembly of the heterodinuclear cofactor in the two different protein groups in which it has been identified, ribonucleotide reductase R2c proteins and R2-like ligand-binding oxidases. Interestingly, although the two groups form the same metal cluster they appear to employ partly different mechanisms to assemble it. In addition, it seems that both the thermodynamics of metal binding and the kinetics of oxygen activation play a role in achieving metal specificity.
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| 38. |
- Griese, Julia J., et al.
(författare)
-
Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein
- 2013
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:43, s. 17189-17194
-
Tidskriftsartikel (refereegranskat)abstract
- Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn-II < Fe-II < Ni-II < Co-II < Cu-II > Zn-II). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds FeII over MnII as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.
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| 39. |
- Griese, Julia J., et al.
(författare)
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Ether cross-link formation in the R2-like ligand-binding oxidase
- 2018
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 23:6, s. 879-886
-
Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of Mn-II and Fe-II in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.
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| 40. |
- Griese, Julia J., et al.
(författare)
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Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion : Q-XAD
- 2019
-
Ingår i: Acta Crystallographica Section D. - 2059-7983. ; D75, s. 764-771
-
Tidskriftsartikel (refereegranskat)abstract
- Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.
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| 41. |
- Griese, Julia J., et al.
(författare)
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Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor
- 2015
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 290:42, s. 25254-25272
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Tidskriftsartikel (refereegranskat)abstract
- Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn-II and Fe-II in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R.M., Lehtio , J., Graslund, A., Lubitz, W., Siegbahn, P. E., and Hogbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 1718917194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.
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| 42. |
- Griese, Julia J., et al.
(författare)
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X ray reduction correlates with soaking accessibility as judged from four non crystallographically related diiron sites
- 2012
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Ingår i: METALLOMICS. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 4:9, s. 894-898
-
Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.
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| 43. |
- Hammerstad, Marta, et al.
(författare)
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The class Ib ribonucleotide reductase from Mycobacterium tuberculosis has two active R2F subunits
- 2014
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 19:6, s. 893-902
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class Ib RNRs require either a diiron-tyrosyl radical (Y center dot) or a dimanganese-Y center dot cofactor in their R2F subunit to initiate ribonucleotide reduction in the R1 subunit. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains two genes, nrdF1 and nrdF2, encoding the small subunits R2F-1 and R2F-2, respectively, where the latter has been thought to serve as the only active small subunit in the M. tuberculosis class Ib RNR. Here, we present evidence for the presence of an active Fe (2) (III) -Y center dot cofactor in the M. tuberculosis RNR R2F-1 small subunit, supported and characterized by UV-vis, X-band electron paramagnetic resonance, and resonance Raman spectroscopy, showing features similar to those for the M. tuberculosis R2F-2-Fe (2) (III) -Y center dot cofactor. We also report enzymatic activity of Fe (2) (III) -R2F-1 when assayed with R1, and suggest that the active M. tuberculosis class Ib RNR can use two different small subunits, R2F-1 and R2F-2, with similar activity.
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| 44. |
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| 45. |
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| 46. |
- Högbom, Martin, et al.
(författare)
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Crystal Structure of Conserved Domains 1 and 2 of the Human DEAD-box Helicase DDX3X in Complex with the Mononucleotide AMP
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 372:1, s. 150-159
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Tidskriftsartikel (refereegranskat)abstract
- DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central β-sheet flanked by α-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPγS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPγS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.
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| 47. |
- Högbom, Martin, et al.
(författare)
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Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes.
- 2002
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Ingår i: Biochemistry. - 0006-2960. ; 41:4, s. 1381-9
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.
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| 48. |
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| 49. |
- Högbom, Martin, et al.
(författare)
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Functional and structural characteristics of bacterial proteins that bind host cytokines
- 2017
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Ingår i: Virulence. - : Informa UK Limited. - 2150-5594 .- 2150-5608. ; 8:8, s. 1592-1601
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Forskningsöversikt (refereegranskat)abstract
- Several human pathogens bind and respond to host cytokines, which can be considered a virulence mechanism that communicates defensive actions of the host to the pathogen. This review summarizes the current knowledge of bacterial cytokine-binding proteins, with a particular focus on their functional and structural characteristics. Many bacterial cytokine-binding proteins function in the development of infection and inflammation and mediate adhesion to host cells, suggesting multiple roles in pathogen-host interactions. The regions of the bacterial proteins that interact with host cytokines can display structural similarities to other proteins involved in cytokine signaling. However, there appears to be no central shared structural themes for bacterial cytokine-binding proteins, and they appear to possess structures that are different from the cytokine receptors of the host. Atomic-level information regarding receptor-cytokine interactions is needed to be able to disrupt these interactions and to elucidate the specific consequences of cytokine binding in a pathogen and host.
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| 50. |
- Högbom, Martin
(författare)
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Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear-an intricate bioinorganic workaround to use different metals for the same reaction
- 2011
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Ingår i: METALLOMICS. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 3:2, s. 110-120
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Forskningsöversikt (refereegranskat)abstract
- The ferritin-like superfamily comprises of several protein groups that utilize dinuclear metal sites for various functions, from iron storage to challenging oxidations of substrates. Ribonucleotide reductase R2 proteins use the metal site for the generation of a free radical required for the reduction of ribonucleotides to deoxyriboinucleotides, the building blocks of DNA. This ubiquitous and essential reaction has been studied for over four decades and the R2 proteins were, until recently, generally believed to employ the same cofactor and mechanism for radical generation. In this reaction, a stable tyrosyl radical is produced following activation and cleavage of molecular oxygen at a dinuclear iron site in the protein. Discoveries in the last few years have now firmly established that the radical generating reaction is not conserved among the R2 proteins but that different subgroups, that are structurally very similar, instead employ di-manganese or heterodinuclear Mn-Fe cofactors as radical generators. This is remarkable considering that the protein must exercise a strict control over oxygen activation, reactive metal-oxygen intermediate species and the resulting redox potential of the produced radical equivalent. Given the differences in redox properties between Mn and Fe, use of a different metal for this reaction requires associated adaptations of the R2 protein scaffold and the activation mechanism. Further analysis of the differences in protein sequence between R2 subgroups have also led to the discovery of new groups of R2-like proteins with completely different functions, expanding the chemical repertoire of the ferritin-like superfamily. This review describes the discoveries leading up to the identification of the different Mn-containing R2 protein groups and our current understanding of them. Hypotheses regarding the biochemical rationale to develop these chemically complex alternative solutions are also discussed.
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